SYMPOSIUM: RESEARCH ADVANCES AFTER A DECADE OF WAR

Porphyrin-adsorbed Allograft Bone: A Photoactive, AntibiofilmSurface

Sana S. Dastgheyb PhD, Cyrus B. Toorkey PhD,

Irving M. Shapiro BDS, PhD, Noreen J. Hickok PhD

Published online: 17 April 2015

� The Association of Bone and Joint Surgeons1 2015

Abstract

Background Allograft bone is commonly used to aug-

ment bone stock. Unfortunately, allograft is prone to

bacterial contamination and current antimicrobial therapies

are inadequate. Photoactivated porphyrins combat bacterial

growth by production of reactive oxygen species (ROS);

however, to our knowledge, they have not been tested in

the setting of allograft bone.

Questions/purposes We asked: (1) Does 5,10,15,20-te-

trakis-(4-aminophenyl)-porphyrin (TAPP) stably adsorb to

morselized, mineralized allograft? (2) Does Staphylococ-

cus aureus acquire TAPP from TAPP-allograft? (3) Is

TAPP-allograft antibacterial to S. aureus? (4) Is ROS

production critical for antimicrobial activity? (5) Does il-

luminated TAPP-allograft dislodge biofilm? (6) Could

other photoactive dyes (TAPP, TMPyP, TSP, THP, and

methylene blue) confer antimicrobial properties to

allograft?

Methods TAPP adsorption to allograft (TAPP-allograft),

its localization in S. aureus, and TAPP-allograft long-term

stability were determined spectrophotometrically. Antimi-

crobial activity was measured while activated with light or

in the dark during incubation with S. aureus or after allo-

graft biofilm formation. Glutathione was added to

illuminated TAPP-allograft to quench ROS and antimi-

crobial activity was determined. Light-dependent

antimicrobial activity of other photoactive dyes (TMPyP,

TSP, THP, and methylene blue) adsorbed to allograft was

also tested.

Results We found (1) porphyrins strongly adhere to bone

allograft; and (2) the bacteria are not able to sequester

TAPP from the TAPP-allograft; (3) when illuminated,

TAPP-allograft is resistant to bacterial adherence; (4) the

effects of TAPP are inhibited by the radical scavenger

glutathione, indicating ROS-dependent antimicrobial ac-

tivity; (5) illumination of TAPP-allograft disrupts biofilms;

and, (6) other photoactive dyes impede biofilm formation

on allograft bone in the presence of light.

Conclusions Porphyrins stably associate with allograft

and are inactive until illuminated. Illuminated TAPP-allo-

graft markedly reduces bacterial colonization, which is

restored in the presence of radical scavengers. Finally, il-

luminated TAPP-allograft disrupts biofilms.

Clinical Relevance The findings of this in vitro study

suggest that loading bone allograft with biocompatible

porphyrins before surgery might allow increased sterility of

the allograft during implantation. Future testing in an ani-

mal model will determine if these in vitro activities can be

One of the authors (SSD) was supported by National Institute of

Arthritis and Musculoskeletal and Skin Diseases training grant T32-

AR-052273 during the study period (USD 10,000 to USD 100,000).

One of the authors (IMS) received research funding from National

Institutes of Health grant R01 HD061053 during the study period

(USD 100,000 to USD 1,000,000). One of the authors (NJH) has

received research funding from NIH grants HD06153 and DE019901

(USD 100,000 to USD 1,000,000), Synergy Biomedical, Collegeville,

PA, USA (USD 10,000 to USD 100,000), and Zimmer, Warsaw, IN,

USA (USD 10,000 to USD 100,000) during the study period. The

Musculoskeletal Transplant Foundation provided human bone

allograft.

All ICMJE Conflict of Interest Forms for authors and Clinical

Orthopaedics and Related Research1 editors and board members are

on file with the publication and can be viewed on request.

Clinical Orthopaedics and Related Research1 neither advocates nor

endorses the use of any treatment, drug, or device. Readers are

encouraged to always seek additional information, including FDA-

approval status, of any drug or device prior to clinical use.

S. S. Dastgheyb, C. B. Toorkey, I. M. Shapiro, N. J. Hickok (&)

Department of Orthopaedic Surgery, Thomas Jefferson

University, 1015 Walnut Street, Suite 501, Philadelphia,

PA 19107, USA

e-mail: Noreen.Hickok@jefferson.edu

123

Clin Orthop Relat Res (2015) 473:2865–2873

DOI 10.1007/s11999-015-4299-5

Clinical Orthopaedicsand Related Research®

A Publication of The Association of Bone and Joint Surgeons®

used to prevent allograft-based infection in an establishing

osteomyelitis.

Introduction

Infection occurs with bone allograft use with rates of ap-

proximately 11% for large allografts and 4% to 6% in hip

revisions [9]. Infection becomes more common when bone

supplementation is used during revision of an infected site

or after ballistic injury. Autologous bone is the gold stan-

dard for replacement and may be associated with a lower

risk of infection than allograft; however, donor site mor-

bidity and poor or inadequate bone stock limit its

availability. Thus, allograft bone remains in wide use.

Allograft-associated infection arises when allograft is

seeded with bacteria from a previous osteomyelitis, a

traumatic injury, or hematogenous bacterial contamination.

The previously planktonic bacteria adhere to the allograft

and initiate production of a biofilm slime, effectively de-

creasing bacterial antibiotic susceptibility [1, 3] and

limiting immune surveillance [25]. Although some antibi-

otics show moderate success in eradicating surface-

associated/biofilm-encased bacteria, the concentrations are

often toxic for the target tissue. Considerable success has

been achieved by adsorption of antibiotics to allograft and

by use of controlled-release bone substitutes [11].

To lessen concerns about fostering antibiotic resistance,

photoactivated compounds are being explored to replace and/

or augment antibiotic treatments. Phototherapy has been used

as an antibacterial treatment in light-accessible sites such as

gingival tissue and dental implantation [22]. We reasoned

that a similar effect could be achieved by illumination during

insertion of allograft into a surgical site. Although the

mechanism is not fully understood, the antibacterial effects of

porphyrins are mostly attributed to their localization in the

bacterial membrane and, when activated by light, generate

reactive oxygen species (ROS) [15]. Because ROS is an-

tibacterial, surface-coupled porphyrins can actively sterilize

surfaces when illuminated [6, 14]. Our laboratory has been

exploring the use of the cationic porphyrin 5,10,15,20-te-

trakis-(4-aminophenyl)-porphyrin (TAPP; Fig. 1) as an

antibacterial treatment for bone allograft.

In this article, we asked: (1) Does TAPP stably adsorb to

morselized, mineralized allograft? (2) Does Staphylococ-

cus aureus extract TAPP from the TAPP-allograft? (3)

Does TAPP-allograft resist colonization by S. aureus? (4)

Is ROS production critical for the antimicrobial activity?

(5) Does illuminated TAPP-allograft dislodge biofilm? (6)

Could other photoactive dyes (ie, TAPP, TMPyP, TSP,

THP, and methylene blue) confer antimicrobial properties

to allograft?

Materials and Methods

Study Design

To test Question 1, if a porphyrin would stably associate

with allograft bone, TAPP (500 lM) was incubated with

allograft overnight, rinsed with phosphate-buffered saline

(PBS), and dried (TAPP-allograft). We measured adsorp-

tion of TAPP to allograft bone (cortical and cancellous

morsels; Musculoskeletal Transplant Foundation, Edison,

NJ, USA) by measuring the decrease in absorbance

(k = 435 nm) of 125 lM TAPP solution before and after

1 day incubation with allograft. TAPP elution from TAPP-

allograft was measured spectrophotometrically after incu-

bation in distilled water over a 9-week period. To measure

the total amount of TAPP adsorbed to the bone, TAPP was

eluted by 5-minute incubation in either 0.275 N HCl or

10 lN HCl.

Because the effects of TAPP are attributed to its in-

corporation into the bacterial membrane, we next asked if

TAPP localized in the bacterial membrane when incubated

with TAPP-allograft (Question 2). Planktonic S. aureus in

PBS were incubated with 200 lM TAPP or with 1 g of

TAPP-allograft overnight. Bacteria were then collected,

washed, lysed, clarified (4000 rpm, 15 minutes), and TAPP

concentration in the lysate was measured spectrophoto-

metrically (TECAN Infinite M1000; Tecan Group,

Mannedorf, Switzerland).

We then tested Question 3: Does TAPP-allograft resist

colonization by S. aureus? A total of 107 CFU/mL S. au-

reus AH1710 (a green-fluorescent protein-expressing strain

[16]) in trypticase soy broth (TSB) was incubated with

TAPP-allograft under illumination (light) or in the dark.

Bacteria adherent to the TAPP-allograft surface were vi-

sualized by confocal laser scanning microscopy or

Fig. 1 Photoactivation of TAPP-adsorbed bone allograft. Bone chips

are incubated with 5,10,15,20-tetrakis-(4-aminophenyl)-porphyrin

(TAPP), structure shown above the arrow to produce TAPP adsorbed

to mineralized bone allograft. When illuminated (hm), excitation of

the TAPP-adsorbed to allograft can produce ROS (O21) in the

immediate vicinity of the surface (green haze); these ROS are known

to be antimicrobial.

2866 Dastgheyb et al. Clinical Orthopaedics and Related Research1

123

recovered by sonication in 0.3% Tween-20 in PBS fol-

lowed by serial dilution/plating.

To test Question 4, if ROS production is critical for the

antimicrobial activity, illuminated control or TAPP-allograft

was incubated with S. aureusAH1710 in the presence of 0 or

20 mM glutathione, a known radical scavenger; adherent

bacteria were visualized by confocal scanning microscopy.

We next tested Question 5: does illuminated TAPP-allo-

graft dislodge biofilm? Biofilms of S. aureus AH1710 were

formed by incubating control or TAPP-allograft for 48 hours

in the dark using TSB containing 1% glucose. Biofilm-

coated control or TAPP-allograft was then placed in the dark

(no activation) or illuminated (light-activated) for 18 hours

in PBS at 37 �C. Biofilm presence was determined by con-

focal microscopy with three-dimensional reconstruction (Z-

stack); alternately, biofilm bacteria were recovered by

sonication in 0.3% Tween-20, diluted, plated, and counted.

Finally (Question 6), we asked if other porphyrins (ca-

tionic: TAPP and meso-tetra[4-sulfonatophenyl] porphine

[TMPyP, 500 lM], anionic: tetrakis[1-methylpyridinium-4-

yl]porphyrin p-Toluenesulfonate [TSP, 500 lM], and neutral:

tetrakis-pyridyl-tetrahydroporphyrin tosylate [THP, 1 mM]),

or the photoactive dye, methylene blue, adsorbed to hydrated,

mineralized, bone allograft to render it antimicrobial. After

overnight adsorption, the allografts were washed and incu-

bated in the dark for 24 hours at 37 �Cwith S. aureusAH1710

in TSB containing 1% glucose to allow biofilm formation.

Biofilm-coated samples were then placed in the light or dark

for 18 hours, rinsed, and adherent bacteria were then visual-

ized with confocal laser scanning microscopy.

Materials

TAPP, TSP, TMPyP, THP, and methylene blue were pur-

chased from Frontier Scientific (Logan, UT, USA).

Glutathione (98%) and 0.4% Trypan blue were purchased

from Sigma-Aldrich Chemical Company (St Louis, MO,

USA). Mineralized bone allograft, cortical and cancellous

granules, 0.5 to 5.0 mm in diameter, was the kind gift of

the Musculoskeletal Transplant Foundation (Edison, NJ,

USA). Vancomycin hydrochloride was obtained from APP

Pharmaceuticals (Lake Zurich, IL, USA); TSB and Bac-

toTM Agar were obtained from Becton, Dickinson, and

Company (Franklin Lakes, NJ, USA). All other materials

were tissue culture grade or analytical grade and were

purchased from Fisher Scientific (Waltham, MA, USA).

Bacterial Culture

S. aureus AH1710, which expresses green fluorescent

protein (GFP) under chloramphenicol pressure [8, 16], was

grown at 37 �C, 225 rpm, in TSB containing 8 lg/mL of

chloramphenicol, 1% glucose. Using a 0.5 McFarland

standard (a measure of light dispersion by suspended par-

ticles in solution [17] that approximates 1 9 108 colony-

forming units [CFU]/mL S. aureus [2]), the culture was

brought to 108 CFU/mL and diluted in PBS as indicated.

Illumination Conditions

All samples to be illuminated were placed in a tissue cul-

ture plate that was then placed approximately 5 inches

below a 100-W, 120-V Sylvania white light (OSRAM,

Danvers, MA, USA) in a humidified, transparent chamber;

distance from the light was slightly adjusted to maintain the

chamber at 37 �C. Controls were incubated in the dark at

37 �C.

Adsorption of Porphyrins to Allograft

Human bone granules (mineralized cancellous and cortical)

were hydrated by washing and sonicating in dH2O until

washings were clear (referred to as allograft). Two milli-

liters of 500 lM TAPP, TMPyP, TSP, methylene blue, or

1 mM THP were incubated overnight at room temperature

with 2 g allograft. All allografts were rinsed with sterile

PBS (three times) before use.

Measurement of Adsorbed TAPP

Allograft (approximately 1 mm3) was added to a 96-well

plate containing 125 lM TAPP (100 lL/well), sealed with

ParafilmTM (Bemis, Oshkosh, WI, USA), and incubated at

room temperature with shaking. At 24 hours, bone morsels

were removed, and absorbance (k = 435 nm [19]) of the

solution was measured. TAPP adsorption to allograft was

calculated by subtracting peak absorbance before and after

allograft incubation and calculating the resulting

concentration.

TAPP-allograft Stability

Allograft morsels soaked in 1 mM TAPP overnight were

washed three times with distilled, deionized water

(ddH2O), and individual morsels were placed into 200 lLddH2O. Samples were maintained at room temperature in

the dark with shaking. Bathing fluid was removed weekly

for 9 weeks and stored in the dark, 4 �C until measure-

ment. At the end of the elution period, remaining TAPP

adsorbed to the allograft was eluted by incubation in 10 lN

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123

HCl or 0.275 N HCl (TAPP is highly soluble at acidic pHs)

for 5 minutes. Absorbance of samples was determined

spectrophotometrically (k = 435 nm) and compared with

100 lL of 125 lM TAPP stored under the same conditions.

Recovery of TAPP from S. aureus

Five milliliters of TSB containing 109 CFU/mL S. aureus

ATCC1 25923TM were incubated in TSB, in 200 lMTAPP, or with 1 g TAPP-allograft for 18 hours at 37 �C.After vortexing, bacteria were pelleted (1500 rpm, 5 min-

utes) and washed in water three times. Pelleted bacteria

were lysed by sonication (Branson 1510; Branson Ultra-

sonics, Danbury, CT, USA) in 200 lL of 0.3% Tween-20

for 20 to 30 minutes and the lysate was analyzed spec-

trophotometrically for porphyrins.

Bacterial Adherence to Bone Allograft

Sterile control or TAPP-allograft morsels were placed in

200 lL PBS containing 107 CFU/mL S. aureus AH1710

and incubated in the light (to activate porphyrin) or dark

(control) at 37 �C for 18 hours. After incubation, morsels

were rinsed three times with PBS and surface colonization

visualized by confocal laser scanning microscopy using

three-dimensional reconstruction.

Addition of Glutathione as an Antioxidant

The effect of glutathione on bacterial growth was deter-

mined using CLSI M31-A2 and glutathione concentrations

of 0 to 80 mM. After overnight incubation at 37 �C, glu-tathione concentrations above 40 mM showed no bacterial

growth. Glutathione (stock: 640 mM GSH in PBS; final

concentration, 20 mM) or PBS was added to wells con-

taining 107 CFU bacteria in the presence of control or

TAPP-allograft (final volume = 200 lL TSB). After light

activation (or no light for control) for 18 hours at 37 �C,samples were washed with PBS and surface colonization

visualized using confocal laser scanning microscopy.

Allograft Biofilm

To form a biofilm, approximately 0.2 g of control or TAPP-

allograft was incubated with 1 mL of 104 CFU/mL S. aureus

AH1710 in TSB containing 1% glucose, 10 lg chloram-

phenicol (to maintain GFP expression) in the dark under static

conditions for 2 days followed by three gentle washes with

PBS. Biofilm-containing morsels were then incubated in the

dark (control) or light (to activate TAPP) at 37 �C for

18 hours. Adherent bacteria were imaged for 50% of samples

using confocal laser scanning microscopy. Adherent bacteria

(other 50% of samples) were also recovered, diluted, plated,

and counted.

Bacterial Survival Counts

Allograft was gently washed 9 3 with PBS followed by

sonication in 200 lL 0.3% Tween-20 for 5 minutes to

suspend surface-adherent bacteria. Suspended bacteria

were diluted in PBS, plated on 3MTM PetrifilmsTM (3M, St

Paul, MN, USA), incubated for 24 hours at 37 �C, andcolonies were counted manually.

Confocal Laser Scanning Microscopy

S. aureus AH1710 was visualized on bone allograft sam-

ples using a 94 lens with 500-lm sections at 25-lmintervals on an Olympus Fluoview 300 confocal micro-

scope (Olympus America Inc, Center Valley, PA, USA).

Three-dimensional images were reconstructed and images

were assessed for number of bacteria (green colonies) as

well as density of colonization and presence of biofilm-like

structures.

Statistical Methods

All experiments were repeated a minimum of three times in

three independent experiments with data expressed as

mean ± SD. One- or two-way analyses of variance, as

appropriate, with a Tukey post hoc multiple comparison

procedure were used to analyze differences between

groups.

Results

Does TAPP Adsorb to Mineralized Allograft?

When morselized, mineralized bone (1 mm3 in 200 lL)was incubated overnight with 125-lM TAPP, the amount

of TAPP remaining in solution decreased to 8 to 10 lMTAPP (Fig. 2A), suggesting adsorption to bone (TAPP-

allograft). Over a 9-week incubation in PBS nor after a 1-

hour incubation in pH 5 water, TAPP-allograft showed no

detectable TAPP elution (Fig. 2B). When TAPP was in-

cubated in a pH 1 solution to allow ready solubilization,

TAPP was completely removed from allograft.

2868 Dastgheyb et al. Clinical Orthopaedics and Related Research1

123

Does S. aureus Extract TAPP from the TAPP-allograft?

TAPP, which can localize to bacterial membranes, showed

an accumulation of approximately 50 lM in planktonic S.

aureus that had been lysed after culturing in 500 lM TAPP

for 18 hours (Fig. 2C) with a slight shift in peak ab-

sorbance. S. aureus cultured on TAPP-allograft, however,

did not appear to sequester TAPP as little absorbance was

measured after lysis.

Does TAPP-allograft Resist Colonization by S. aureus?

Control allograft was abundantly colonized in both the dark

and the light; S. aureus also colonized TAPP-allograft in

the dark. In contrast, illuminated TAPP-allograft showed

little bacterial colonization (Fig. 3A). When adherent

bacteria were recovered and plated, similar numbers of S.

aureus (105–106 CFU/mL) were measured on control al-

lograft (light and dark) and dark TAPP-allograft; no viable

bacteria were recovered from TAPP-allograft that had been

incubated with S. aureus in the light (Fig. 3B).

Is ROS Production Critical for Antimicrobial Activity?

In the presence of 0 or 20 mM glutathione, a radical

scavenger that will inactivate ROS, S. aureus readily

colonized both dark and light control (PBS) allograft.

Minimal colonization was present on illuminated TAPP-

allograft without glutathione, but in the presence of 20 mM

glutathione, some colonization was regained (Fig. 3C).

Does Illuminated TAPP-allograft Dislodge Biofilm?

S. aureus biofilms (48 hours, dark) were formed on TAPP

and control allograft. Control/PBS allograft remained

abundantly colonized after an additional 18 hours in the

light or dark as did dark TAPP-allograft. In contrast, bio-

film bacteria were not obvious on the illuminated TAPP-

allograft samples (Fig. 4A). Numbers of adherent bacteria

recovered from PBS allograft (light and dark) and dark

TAPP-allograft samples were approximately equivalent;

illuminated TAPP-allograft samples showed a 2- to 3-log

decrease in adherent bacteria (Fig. 4B).

Other Photosensitizers on Allograft

In the dark, S. aureus colonized all allografts with adsorbed

photosensitizers (cationic porphyrins TAPP or TMPyP,

anionic porphyrin TSP, neutral porphyrin THP, or methy-

lene blue). After illumination, with the exception of

methylene blue, all photosensitizer allograft samples

showed decreased bacterial colonization. Methylene blue

did not remain adsorbed to allograft so was unable to de-

crease bacterial colonization (Fig. 5).

Discussion

Bone allograft is an important material for bone augmen-

tation but, like any other implanted material, is prone to

Fig. 2A–C TAPP stably adsorbs to mineralized bone allograft.

(A) Absorbance spectra of TAPP before (125 lM, solid line) and

after (TAPP remaining, dashed line) 24-hour incubation at room

temperature with bone morsels. (B) Absorbance of solutions that werecollected weekly during elution from TAPP-allograft for up to

9 weeks or after exposure to 10 lN (pH 5) or 0.275 N (pH 1) HCl.

Values shown are mean ± SD. The graph is a representative

experiment with triplicate samples; the experiment was performed a

total of three times with similar results. (C) TAPP absorbance was

measured in solution (black line). Absorbance of TAPP localized in

planktonic S. aureus was measured after incubation (18 hours),

washing, and lysis (gray line); and TAPP localized in S. aureus was

measured after incubation (18 hours) with TAPP-allograft followed

by washing and lysis (broken line).

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123

bacterial colonization. We asked if photoactivated com-

pounds adsorbed to these allografts could confer

antimicrobial activity to allograft while illuminated. Pre-

vious work has shown that photoactivation of porphyrin in

solution kills planktonic bacteria [8] and to some extent

biofilms [5]. We show here that porphyrins adsorb avidly to

mineralized allograft. This adsorption is stable over

9 weeks. Furthermore, S. aureus does not solubilize TAPP

from the bone matrix. In the dark, S. aureus readily adheres

and grows in the presence of porphyrins. However, light

activation results in rapid killing. This killing is presum-

ably the result of the production of ROS as a radical

scavenger (glutathione) preventing this effect. Light acti-

vation of the TAPP-allograft can cause eradication of

biofilms resident on the surface, and these activities are not

unique to TAPP because other porphyrins can cause similar

effects.

The significance of these findings resides in the as-

sumption that many surgical site infections are initiated

during the operation. In the bright environment of the

operating room, TAPP-allograft could be illuminated by

exposure to intense white light and this illumination

would then continue during insertion, ensuring that the

implants were sterile. However, these conditions need to

be explicitly tested because the work that we describe in

this article has been limited to standard bacterial growth

media and TAPP-allograft has not been tested in the

presence of physiological fluids. Additionally, only

morselized allograft, not the widely used large, structural

allograft, was tested, although allograft morsels contain

both cancellous and cortical bone. Although application

to the large allografts may be possible, it needs to be

explicitly tested, especially because the geometry of the

larger allografts may constrain even illumination. Finally,

we used a broad-spectrum lamp rather than a targeted

wavelength and we consistently photoactivated for ap-

proximately 18 hours. Use of a targeted wavelength and

defining the minimum photoactivation time could allow

more efficient photoactivation for maintenance of

sterility.

We first investigated if TAPP adsorbed to mineralized

allograft. Our data showing that porphyrins avidly ad-

sorbed to mineralized bone allograft are consistent with

previous descriptions of the accumulation of porphyrins in

mineralized tissue and their strong affinity for teeth and

calcified plaques in the circulatory system [12]. This ad-

sorption could only be disrupted by strong acidification, as

we observed in our studies. Practically, porphyrins accu-

mulate in bone with no clearance until bone/tissue turnover

has occurred [10, 20].

Because the antibacterial activity of porphyrins is as-

sociated with their localization in cells [4], we next asked if

bacteria were able to extract TAPP from TAPP-allograft.

Although S. aureus readily acquired TAPP from solution,

minimal TAPP was detected in bacteria incubated with

TAPP-allograft. These results suggest that bacteria will

have to come in contact with the TAPP-allograft for its

antibacterial effects.

Next, we addressed if TAPP-allograft resists coloniza-

tion by S. aureus under illumination. We showed that

Fig. 3A–C Illuminated TAPP-allograft is antibacterial and its ac-

tivity is moderated by glutathione. (A) Control allograft (PBS) or

TAPP-allograft was incubated (18 hours) with S. aureus in the dark or

while illuminated. Adherent bacteria (green) were visualized by

confocal laser scanning microscopy. (B) Number of adherent bacteria

recovered from the different allograft surfaces as determined by direct

counting. The table indicates the absence (�) or presence (+) of light

activation (Illumination), TAPP adsorbed to allograft (TAPP), and the

presence of allograft bone (Allograft). **p\ 0.01 when compared

with the dark control as determined by one-way analysis of variance.

(C) Control (PBS) or TAPP-allograft was incubated with S. aureus in

the light in the presence of 0 or 20 mM glutathione (GSH). Bacterial

adherence and density (green) was visualized by confocal laser

scanning microscopy. For A and C, magnification: scale

bar = 500 lm.

2870 Dastgheyb et al. Clinical Orthopaedics and Related Research1

123

TAPP-allograft, when illuminated, robustly decreased

bacterial colonization. This effect was observed during

incubation with planktonic bacteria, a situation that would

more nearly recapitulate the perioperative contamination of

allograft. This marked (approximately 5–6 logs) decrease

is high enough to suggest therapeutic efficacy.

We then asked whether ROS production is critical for

the antimicrobial activity. Porphyrins, when internalized in

cells, depend on the production of ROS for their antimi-

crobial activity [8]. Glutathione is a known radical

scavenger that can inhibit the activity of TAPP in solution

[8]. As observed with solution TAPP, glutathione de-

creased the activity of photoactivated TAPP-allograft,

suggesting that ROS at the surface of the allograft mediates

the effect. Furthermore, these data suggest that the an-

timicrobial effects would be localized to the bone-liquid

interface as the mean free path of ROS is on the order of

microns.

We next asked if illuminated TAPP-allograft can dislodge

biofilm. Our results showed that activation of the TAPP-

allograft by illumination causes dispersion of the biofilm

bacteria from the surface. Because of the antibiotic recalci-

trance of adherent/biofilm-associated bacteria [21], biofilms

are often not susceptible to antibiotic levels that are 9 100

to 9 1000 the minimum inhibitory concentration of plank-

tonic bacteria [3, 7]. Biofilms of Porphyromonas gingivalis

have reportedly been treated by light activation of endoge-

nous porphyrin by dentists since 2003 [13, 23]. Therefore,

even in the case in which an allograft has been prepopulated

by bacteria, an effect such as we describe could effectively

sterilize the material before or during the operative

procedure.

Finally, we asked if other photoactive dyes, ie, TAPP,

TMPyP, TSP, THP, and methylene blue, were able to

confer antimicrobial properties to allograft. Based on our

data, the ability of the compound to adsorb to a bone al-

lograft is the most important factor. Whereas all porphyrin

compounds adsorbed avidly to bone, methylene blue, a

strong radical producer, easily eluted from bone because of

its solubility. In keeping with this, the porphyrin allografts

showed antimicrobial activity on illumination, whereas

methylene blue allograft showed very little antimicrobial

activity. Despite this strong adsorption to allograft, charged

porphyrins (ie, TAPP, TMPyP, and TSP) outperformed the

neutral porphyrin THP on illumination. These data are

supported by other reports claiming that both cationic and

anionic photoporphyrins have greater antimicrobial effects

than neutral porphyrins [18, 24]. Taken together, these data

suggest that charged porphyrins, whether anionic or ca-

tionic, would be excellent candidates for photodynamic

antimicrobial therapy. Further studies must be performed to

determine the stability, cytotoxicity, and photodegradation/

photoactivity profiles for each photoactive dye.

In summary, porphyrins are biologically well tolerated

and easily adsorbed to bone, which allows for the option of

creating antimicrobial allografts to combat perioperative

infections. Sterility of biomaterials in the perioperative

period is extremely important to achieve low postoperative

infection rates. The most important application may be the

sterilization of structural allografts, which will require

further development to prove utility. An additional area

that may prove to be of great importance is the possibility

of increasing the efficacy of traditional antibiotics. In fact,

combining the proven efficacy of adsorbed antibiotics with

photoactivated TAPP-allograft could prove to be a potent

new treatment. We propose that use of a biocompatible,

porphyrin-treated bone allograft that may be activated

Fig. 4A–B Illuminated TAPP-allograft disperses biofilm. (A) S.

aureus AH1710 was allowed to form a biofilm on PBS or TAPP-

allograft by incubation for 48 hours in the dark. Bacterial colonization

(as a result of expression of GFP) was visualized after another

18 hours in the dark or after illumination (‘‘a’’ is PBS allograft in the

dark or ‘‘b’’ light; ‘‘c’’ is TAPP allograft in the dark or ‘‘d’’ light).

Magnification: scale bar = 500 lm. (B) Adherent S. aureus were

recovered and counted (shown as CFU/ml) from PBS or TAPP-

allograft treated as in Fig. 4A. *p\ 0.05 when compared with the

other counts as determined by a two-tailed t-test.

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123

under visible light in the perioperative setting may promote

sterility and/or disinfection of the allograft before im-

plantation to ultimately decrease infection rates.

Acknowledgments We thank the Musculoskeletal Transplant

Foundation for supplying bone allograft and Dr Alexander Horswill,

University of Iowa, for allowing us to use his S. aureusAH1710 strain.

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